Method for producing transgenic animals

ABSTRACT

The present invention relates to methods for producing transgenic animals using retroviral constructs engineered to carry a transgene(s) of interest.

REFERENCE TO RELATED APPLICATIONS

The present application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Application No. 60/322,031, filed Sep. 13, 2001 and to U.S.Provisional Application No. 60/347,782, filed Jan. 9, 2002.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant NumberGM39458 awarded by the National Institutes of Health. The United StatesGovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods for generating transgenicanimals using viral constructs engineered to carry the transgene(s) ofinterest.

2. Description of the Related Art

Early transgenic experiments used an oncoretrovirus to introduce thegene of interest into embryonic cells (Jaenisch Proc. Natl. Acad. Sci.USA 73:1260-1264 (1976)). In a typical experiment an engineered Moloneystrain of mouse leukemia virus (MOMLV) was injected into the blastocystcavity of mice. While the transgene was often integrated into the genomeof the resulting mice, no gene expression could be detected.

Today, the majority of transgenic animals are made using directinjection technology (Gordon and Ruddle Science 214:1244-1246 (1981)).Briefly, a DNA construct carrying the gene of interest is injecteddirectly into the pronucleus of a single-cell zygote. The cell is thenimplanted into a pseudo-pregnant female and the resulting progeny isanalyzed for expression of the gene.

While this method achieves both integration and expression of thetransgene, there are a number of significant drawbacks to the directinjection technique. First, in order to carry out the technique it isnecessary to inject DNA directly into the pronucleus. This is possiblein some specific strains of mice, such as Black6×BDA, because the malepronucleus is visible. However, in other animals and other strains ofmice the pronucleus is less visible, making the technique extremelydifficult. Further, the injection requires the assistance of a skilledtechnician and a significant investment in equipment; micromanipulatorsare necessary to hold the cell and the injection pipette and a pressuresource is required that can deliver picoliter amounts of DNA solution.

A second equally significant problem with the direct injection method isthe low percentage of injected zygotes that produce transgenic animals.The injection pipette must go through the zona pellucida, the cellmembrane and the nuclear envelope. Thus only 80-90% of mouse cellssurvive the injection. Other animal cells are less hardy and thesurvival rates are somewhat lower, with about 60% survival for rats and40-50% for cows. In mice, of the original zygotes, about 25% aresuccessfully injected and implanted in a pseudopregnant female. About20% of the resulting animals have the transgene integrated into theirgenome. Of these, about 20% will express the gene. However, even if theanimals express the gene, it is possible that the expression patternwill not be useful. Thus, only about 1% of injected zygotes result intransgenic animals that express the gene of interest. This lowefficiency of transgenesis is particularly troubling for larger animals,such as pigs, cows or goats, in which obtaining large numbers of embryosis not possible (see, e.g., Wall et al. J. Cell. Biochem. 49:113(1992)).

In addition, direct pronuclear injection is not possible for many typesof animals, including birds.

SUMMARY OF THE INVENTION

One aspect of the present invention concerns a method of producing atransgenic animal. In the preferred embodiment a packaging cell line istransfected with a retroviral construct, recombinant retrovirus iscollected, and an embryonic cell is infected with the recombinantretrovirus. The retroviral construct preferably comprises the R and U5sequences from a 5′ lentiviral long terminal repeat (LTR) and aself-inactivating lentiviral 3′ LTR. Further, the self-inactivating 3′LTR preferably comprises a U3 element with a deletion of its enhancersequence. In one embodiment the LTR sequences are from HIV.

In one embodiment the retroviral construct comprises a gene that isdesirably expressed in the transgenic animal. In this embodiment theretroviral construct may also comprise an internal promoter and/orenhancer. In one embodiment the internal promoter is ubiquitous. Theubiquitous promoter may be selected from the group consisting of theubiquitin promoter, the CMV β-actin promoter and the pgk promoter. Inanother embodiment the internal promoter is tissue-specific. The tissuespecific promoter may be selected from the group consisting of the lckpromoter, the myogenin promoter and the thy1 promoter.

In addition, the recombinant retrovirus may be pseudotyped. Thus, in oneembodiment the recombinant retrovirus is pseudotyped with the vesicularstomatitis virus envelope glycoprotein. In another embodiment therecombinant retrovirus is pseudotyped with a mutant ecotropic envelopeprotein, preferably ecotropic envelope protein 4.17.

The viral construct may comprise one or more additional geneticelements. In one embodiment the viral construct comprises a promoteroperably linked to the R and U5 5′ LTR sequences, preferably a CMVpromoter sequence. An enhancer, preferably a CMV enhancer sequence, mayalso be included in the viral construct.

In another embodiment the viral construct comprises a woodchuckhepatitis virus enhancer element sequence. In yet another embodiment theviral construct comprises a tRNA amber suppressor sequence.

The viral construct may additionally comprise a reporter gene operablylinked to the internal promoter. The reporter gene may encode be afluorescent protein, preferably green fluorescent protein.

In one embodiment the embryonic cell is infected by injecting therecombinant retrovirus between the zona pellucida and the cell membrane.In another embodiment the embryonic cell is infected by removing thezona pellucida, preferably be enzymatic digestion, and incubating thedenuded cell in a solution containing the recombinant retrovirus.

In another aspect the invention concerns a method of producing atransgenic mammal comprising removing the zona pellucida from anembryonic cell, contacting the embryonic cell with a modified retrovirusand implanting the embryonic cell in a pseudopregnant female. Theembryonic cell is preferably a one-cell embryo. The retrovirus ispreferably a modified lentivirus. The modified lentivirus is preferablyproduced by transfecting a packaging cell line with a viral construct.The viral construct may comprise the R and U5 sequences from alentiviral 5′ LTR, an internal promoter, a gene of interest and a selfinactivating lentiviral 3′ LTR.

In another aspect the invention concerns a method of producing atransgenic animal comprising injecting a modified lentivirus into theperivitelline space of an embryonic cell.

In yet another aspect the invention concerns a transgenic animal made byany of the disclosed methods. Thus the transgenic animal preferably hasa genome that comprises proviral DNA. The proviral DNA may comprise aself-inactivating lentiviral 3′ LTR, such as a self-inactivating HIV 3′LTR. In particular, the self-inactivating 3′ LTR may have a deletion ofits enhancer sequence.

In a further aspect the invention concerns a viral construct comprisingCMV enhancer/promoter sequences, the R and U5 sequences from the 5′ HIVLTR, the HIV′1 flap signal, an internal enhancer and/or promoter, anexogenous gene of interest, the woodchuck hepatitis virus responsiveelement, a tRNA amber suppressor sequence, a U3 element with a deletionof its enhancer sequence, the chicken β-globin insulator and the R andU5 sequences of the 3′ HIV LTR.

In still further aspects the invention concerns methods of makingtransgenic birds, fish and other animals. In one embodiment a transgenicbird is produced by a method comprising transfecting a packaging cellline with a viral construct, recovering recombinant retroviralparticles, and infecting a bird egg with the recombinant retroviralparticles. Preferably the viral construct comprises the R and U5sequences from a 5′ lentiviral LTR and a self-inactivating 3′ lentiviralLTR. Infecting the bird egg preferably comprises contacting theembryonic blastodisc of the bird egg with the retroviral particles.

In another embodiment a transgenic fish is produced by a method thatcomprises transfecting a packaging cell line with a viral construct,recovering recombinant retroviral particles, and infecting a fish eggwith the recombinant retroviral particles. Preferably the viralconstruct comprises the R and U5 sequences from a 5′ lentiviral LTR anda self-inactivating 3′ lentiviral LTR. Infecting the fish egg preferablycomprises delivering the retroviral particles to the space between thechorion and the cell membrane of the fish egg.

In another aspect the invention concerns methods of isolating genesexpressed in particular tissues. In one embodiment embryonic cells areinfected with a modified retrovirus whose genome comprises aself-inactivating lentiviral 3′ LTR, a splice acceptor sequence and areporter gene. Tissues from the developing embryo or mature mammal thatexpress the reporter gene are identified and the gene sequences thatflank the provirus are identified.

In a further aspect the invention concerns methods of identifying genesthat play a role in biological processes. In one embodiment embryoniccells are infected with a modified retrovirus whose genome comprises aself-inactivating lentiviral 3′ LTR. A phenotype in the resultingtransgenic animal is observed and the gene that was disrupted by theprovirus is identified by sequencing the nucleic acid flanking theprovirus.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A is a diagram of the FUGW viral construct. FIG. 1B is a diagramof the provirus that is integrated into the host genome after infectionwith recombinant virus prepared with the FUGW viral construct of FIG.1A.

FIG. 2 is a Southern blot analysis of proviral transgene insertions inthe founder generation of mice generated by injecting recombinantlentivirus into the perivitelline space of one-cell embryos. Genomic DNAfrom each animal was digested with either PstI (left) or BamiHI (right),and probed with a GFP+WRE sequence. All PstI and BamHI sites in theprovirus are located 5′ to the GFP gene. Plus signs above each laneindicate GFP expression in the animal detectable by viewing underconventional epifluorescence.

FIG. 3 is a Southern blot analysis of proviral transgene insertions inthe founder generation of a second group of mice generated by injectingrecombinant lentivirus into the perivitelline space of one-cell embryos.Genomic DNA from each animal was digested with BamHI and probed with aGFP+WRE sequence. All BamHI sites in the provirus are located 5′ to theGFP gene. Plus signs above each lane indicate GFP expression in theanimal detectable by viewing under conventional fluorescence. Of the 56founder animals in this experiment, 45 or 80.4% are transgenic. Of these45 transgenic animals, 41 or 91.1% express GFP at detectable levels.Lanes marked “C” are positive plasmid controls.

FIG. 4 shows ubiquitous GFP expression in rats derived from the deliveryof modified lentivirus to single-cell embryos in vitro. FIG. 4A showsbrightfield (BF) and fluorescent images of the paws of newborn ratsderived from a FUGW-injected embryo. Pup R4, carrying 4 copies of theproviral insert, expresses GFP in the paw, as well as all other tissuesand organs examined. A littermate (R3) carrying no transgene is includedfor comparison. FIG. 4B shows a Southern blot analysis of proviralinsertions in rats generated by injection of FUGW lentivirus into theperivitelline space of single-cell embryos. Genomic DNA was digestedwith PstI and hybridized with a GFP+WRE probe. Plus signs above eachlane indicate GFP expression in the animal detectable by direct viewingunder a fluorescent microscope.

FIG. 5 is a Southern blot analysis of proviral transgene insertions inthe founder generation of mice generated by incubating denuded embryosin media comprising recombinant lentivirus produced with the FUGW viralconstruct. Genomic DNA from each animal was digested with PstI andprobed with a GFP+WRE sequence. All PstI sites in the provirus arelocated 5′ to the GFP gene. The ratios above the lanes indicate thedilution of the virus from 1×10⁶ pfu/μl.

FIG. 6 shows GFP expression in major tissues and organs of a foundermouse. The mouse was perfused intracardially with PBS, pH 7.4, and then3% paraformaldehyde, and viewed immediately under a fluorescentdissecting microscope. The particular mouse shown was generated byco-incubation of the denuded embryo with the lentiviral suspension andcontains 8 proviral insertions. A wildtype animal, identically perfusedand photographed is included for comparison.

FIG. 7 is a Southern blot analysis of proviral transgene insertions inthe F1 progeny of founder transgenic mice, showing that the F1 progenyinherit the proviral transgene in a Mendelian fashion. The founder micewere generated by injection of FUGW lentivirus into the perivitellinespace of single-cell embryos. Genomic DNA from each animal was digestedwith BamHI and probed with GFP+WRE sequence. All BamHI sites are located5′ to the GFP gene. The first numbered lane in each group is the P0founder animal, while the lettered lanes represent progeny resultingfrom outcrossing that founder animal to a wildtype animal. Plus signsabove each lane indicate GFP expression in that animal detectable bydirect viewing of the live animal under a conventional epifluoresencemicroscope.

FIG. 8 shows that transgenic mice give rise to transgenic progeny thatexpress the transgene. This indicates that the transgene can go throughan entire round of gametogenesis and development without being silenced.Expression of the transgene was determined based on GFP expression inthe newborn pup. The pup imaged here is descended from an animal with 10proviral insertions.

FIG. 9 is a Southern blot analysis of proviral transgene insertions inthe founder generation of mice generated using a lentiviral vectorcontaining the myogenin promoter driving a histone2B-GFP fusion. Embryoswere recovered from the uterus at embryonic day 11.5 (“E11.5”). Thelitter consisted of 6 animals, all of which were transgenic. Genomic DNAfrom each animal was digested with BamHI and probed with a GFP+WREsequence. A BamHI site is located within the histone2B gene, 5′ of theGFP sequence in the provirus. Plus signs above the lanes indicatepositive plasmid controls. Animals 5 and 6 were positive fortissue-specific GFP expression at embryonic day 11.5 when viewed as awhole mount under an inverted fluorescent microscope, and animal 5expressed more highly than animal 6.

FIG. 10 shows the GFP expression pattern in an E11.5 mouse embryoderived from the perivitelline space injection of lentivirus carrying ahistone2B-GFP fusion construct under the control of the myogeninpromoter (Yee et al. Genes and Dev. 6:1277-1289 (1993)). GFP expressionis localized to the somites and can be seen in the emerging muscles inthe limb buds, eye and jaw.

FIG. 11 shows immunofluorescence with an antibody against GFP in across-section through an E11.5 embryo carrying the myogenin promoterdriving GFP expression. Embryos were derived from single-cell zygotesinjected with recombinant lentivirus in the perivitelline space. Embryoswere fixed in 3% paraformaldehyde, cryoprotected in 30% sucroseovernight and 30 μm sections were cut on a cryostat. Sections wereincubated with a polyclonal antibody against GFP and probed withα-rabbit secondary antibody conjugated to a rhodamine fluorophore.Images on the left are sections as viewed under a rhodamine filter,while images on the right show the nuclear counterstain Hoechst-33342for each corresponding section. The animal carried 6 proviral insertionsof the myogenin-GFP construct. Specific staining of somite tissues canbe seen, with the exclusion of the stain from flanking skin and bonetissues.

FIG. 12 also shows immunofluorescence with an antibody against GFP incross sections of an E11.5 embryo carrying a myogenin promoter drivingH2B-GFP. Lack of staining in the viscera is noteworthy.

FIG. 13 also shows immunofluorescence with an antibody against GFP incross sections of an E11.5 embryo carrying a myogenin promoter drivingH2B-GFP. Specific staining of somites on either side of the neural tubecan be visualized.

FIG. 14 shows H2B-GFP expression in the extraembryonic tissue ofdeveloping zebra finch.

FIG. 15 shows H2B-GFP expression inside of the zebra finch embryo.

FIG. 16 shows the nucleotide sequence of GFP (SEQ ID NO:3).

FIG. 17 shows the nucleotide sequence of H2B-GFP (SEQ ID NO:5).

FIG. 18A shows the nucleotide sequence of HIV NL4.3 flap (SEQ ID NO:1)and 18B shows the nucleotide sequence of WRE (SEQ ID NO:4).

FIG. 19A shows the nucleotide sequence of the myogenin promoter (SEQ IDNO:7) and 19B shows a partial nucleotide sequence of the Lck promoter(SEQ ID NO:6).

FIG. 20 shows the nucleotide sequence of the human ubiquitin promoter(SEQ ID NO:2).

FIG. 21 shows the nucleotide sequence of the HIV-1flap+ubiquitin+GFP+WRE construct (SEQ ID NO:8).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Retroviruses are enveloped RNA viruses that are capable of infectinganimal cells. When a retrovirus infects a cell, its RNA genome isconverted into a double-stranded linear DNA form by reversetranscription. The DNA form is integrated into the host cell genome as aprovirus. The present invention is based on the discovery thatrecombinant retroviruses can be used to create transgenic animals.Transgenic animals resulting from the methods of the present inventionhave one or more copies of the transgene of interest integrated in theirgenome.

Previous transgenic technology is not commercially practical in largeranimals, such as monkeys, dogs, poultry, cows, pigs or sheep.Furthermore, previous transgenic methods are not applicable to poultry.Thus, the methods of the present invention will find great commercialapplication, for example in biotechnology and agriculture. The presentmethods may be used to introduce the gene of choice into animals inorder to confer upon them desired attributes. For example, the describedmethods may be used to confer disease resistance. In biotechnology, theability to rapidly develop large numbers of transgenic animals,particular higher order animals such as monkeys, will allow for theanalysis of gene function and the evaluation of compounds thatpotentially modulate gene expression, protein function, or are useful intreating a disease or disorder. Two types of assays in which the methodsof the present invention are particularly useful are gene trap assaysand large-scale mutagenesis screens, each of which is described below.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Any methods, devices andmaterials similar or equivalent to those described herein can be used inthe practice of this invention.

By “transgene” is meant any nucleotide or DNA sequence that isintegrated into one or more chromosomes of a host cell by humanintervention, such as by the methods of the present invention. In oneembodiment the transgene comprises a “gene of interest.” A “gene ofinterest” is a nucleic acid sequence that encodes a protein or othermolecule that is desirable for integration and/or expression in a hostcell. In this embodiment the gene of interest is generally operativelylinked to other sequences that are useful for obtaining the desiredexpression of the gene of interest, such as transcriptional regulatorysequences. In another embodiment the transgene can be a DNA sequencethat is used to mark the chromosome where it has integrated. In thissituation, the transgene does not have to comprise a gene that encodes aprotein that can be expressed. This use of the transgene as a moleculartag has numerous applications, for example for mutagenesis studies asdescribed below.

The term “transgenic” is used herein to describe the property ofharboring a transgene. For instance, a “transgenic organism” is anyanimal, including mammals, fish, birds and amphibians, in which one ormore of the cells of the animal contain nucleic acid introduced by wayof human intervention, such as by the methods described herein. In thetypical transgenic animal, the transgene causes the cell to express oroverexpress a recombinant protein. However for some applications, suchas the mutagenesis studies described below, it is not necessary ordesirable for the transgenic organism to express a recombinant protein.

The terms “founder,” “founder animal” and “founder line” refer to thoseanimals that are mature products of the embryos or oocytes to which thetransgene was added, i.e. those animals that grew from the embryos oroocytes into which DNA was inserted.

The terms “progeny” and “progeny of the transgenic animal” refer to anyand all offspring of every generation subsequent to the originallytransformed animal.

The term “animal” is used in its broadest sense and refers to allanimals including mammals, birds, fish, reptiles and amphibians.

The term “mammal” refers to all members of the class Mammalia andincludes any animal classified as a mammal, including humans, domesticand farm animals, and zoo, sports or pet animals, such as mouse, rabbit,pig, sheep, goat, cattle and higher primates.

The term “oocyte” refers to a female gamete cell and includes primaryoocytes, secondary oocytes and mature, unfertilized ovum. As usedherein, the term “egg” when used in reference to a mammalian egg, meansan oocyte surrounded by a zona pellucida. The term “zygote” refers to afertilized ovum. The term “embryo” broadly refers to an animal in theearly stages of development.

“Perivitelline space” refers to the space located between the zonapellucida and the cell membrane of a mammalian egg or embryonic cell.

“Target cell” or “host cell” means a cell that is to be transformedusing the methods and compositions of the invention.

“Lentivirus” refers to a genus of retroviruses that are capable ofinfecting dividing and non-dividing cells. Several examples oflentiviruses include HIV (human immunodeficiency virus; including HIVtype 1, and HIV type 2), the etiologic agent of the human acquiredimmunodeficiency syndrome (AIDS); visna-maedi, which causes encephalitis(visna) or pneumonia (maedi) in sheep, the caprinearthritis-encephalitis virus, which causes immune deficiency, arthritis,and encephalopathy in goats; equine infectious anemia virus, whichcauses autoimmune hemolytic anemia, and encephalopathy in horses; felineimmunodeficiency virus (FIV), which causes immune deficiency in cats;bovine immune deficiency virus (BIV), which causes lymphadenopathy,lymphocytosis, and possibly central nervous system infection in cattle;and simian immunodeficiency virus (SIV), which cause immune deficiencyand encephalopathy in sub-human primates.

A lentiviral genome is generally organized into a 5′ long terminalrepeat (LTR), the gag gene, the pol gene, the env gene, the accessorygenes (nef, vif, vpr, vpu) and a 3′ LTR. The viral LTR is divided intothree regions called U3, R and U5. The U3 region contains the enhancerand promoter elements. The U5 region contains the polyadenylationsignals. The R (repeat) region separates the U3 and U5 regions andtranscribed sequences of the R region appear at both the 5′ and 3′ endsof the viral RNA. See, for example, “RNA Viruses: A Practical Approach”(Alan J. Cann, Ed., Oxford University Press, (2000)), 0 Narayan andClements J. Gen. Virology 70:1617-1639 (1989), Fields et al. FundamentalVirology Raven Press. (1990), Miyoshi H, Blomer U, Takahashi M, Gage FH, Verma I M. J Virol. 72(10):8150-7 (1998), U.S. Pat. No. 6,013,516.

“Virion,” “viral particle” and “retroviral particle” are used herein torefer to a single virus comprising an RNA genome, pol gene derivedproteins, gag gene derived proteins and a lipid bilayer displaying anenvelope (glyco)protein. The RNA genome is usually a recombinant RNAgenome and thus may contain an RNA sequence that is exogenous to thenative viral genome. The RNA genome may also comprise a defectiveendogenous viral sequence.

A “pseudotyped” retrovirus is a retroviral particle having an envelopeprotein that is from a virus other than the virus from which the RNAgenome is derived. The envelope protein may be from a differentretrovirus or from a non-retroviral virus. A preferred envelope proteinis the vesicular stomatitius virus G (VSV G) protein. However, toeliminate the possibility of human infection, viruses can alternativelybe pseudotyped with ecotropic envelope protein that limit infection to aspecific species, such as mice or birds.

The term “provirus” is used to refer to a duplex DNA sequence present ina eukaryotic chromosome that corresponds to the genome of an RNAretrovirus. The provirus may be transmitted from one cell generation tothe next without causing lysis or destruction of the host cell.

A “self-inactivating 3′ LTR” is a 3′ long terminal repeat (LTR) thatcontains a mutation, substitution or deletion that prevents the LTRsequences from driving expression of a downstream gene. A copy of the U3region from the 3′ LTR acts as a template for the generation of bothLTR's in the integrated provirus. Thus, when the 3′ LTR with aninactivating deletion or mutation integrates as the 5′ LTR of theprovirus, no transcription from the 5′ LTR is possible. This eliminatescompetition between the viral enhancer/promoter and any internalenhancer/promoter. Self-inactivating 3′ LTRs are described, for example,in Zufferey et al. J. Virol. 72:9873-9880 (1998), Miyoshi et al. J.Virol. 72:8150-8157 and Iwakuma et al. Virology 261:120-132 (1999).

In one aspect of the invention, a recombinant retrovirus is used todeliver a transgene of interest to a cell, preferably an oocyte or anembryonic cell, more preferably a one-cell embryo. The transgene, andany associated genetic elements, are thus integrated into the genome ofthe host cell as a provirus. The cell may then be allowed to developinto a transgenic animal.

The recombinant retrovirus used to deliver the transgene is preferably amodified lentivirus, and thus is able to infect both dividing andnon-dividing cells. The recombinant retrovirus preferably comprises amodified lentiviral genome that includes the transgene. Further, themodified lentiviral genome preferably lacks endogenous genes forproteins required for viral replication, thus preventing replication inthe transgenic animal. The required proteins are provided in trans inthe packaging cell line during production of the recombinant retrovirus,as described below.

In the preferred embodiment the transgene is incorporated into a viralconstruct that comprises an intact retroviral 5′ LTR and aself-inactivating 3′ LTR. The viral construct is preferably introducedinto a packaging cell line that packages viral genomic RNA based on theviral construct into viral particles with the desired host specificity.Viral particles are collected and allowed to infect the host cell. Eachof these aspects is described in detail below.

The Viral Construct

The viral construct is a nucleotide sequence that comprises sequencesnecessary for the production of recombinant retrovirus in a packagingcell. In one embodiment the viral construct additionally comprisesgenetic elements that allow for the desired expression of a gene ofinterest in the host.

Generation of the viral construct can be accomplished using any suitablegenetic engineering techniques well known in the art, including, withoutlimitation, the standard techniques of restriction endonucleasedigestion, ligation, transformation, plasmid purification, and DNAsequencing, for example as described in Sambrook et al. (MolecularCloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, N.Y.(1989)), Coffin et al. (Retroviruses. Cold Spring Harbor LaboratoryPress, N.Y. (1997)) and “RNA Viruses: A Practical Approach” (Alan J.Cann, Ed., Oxford University Press, (2000)).

The viral construct may incorporate sequences from the genome of anyknown organism. The sequences may be incorporated in their native formor may be modified in any way. For example, the sequences may compriseinsertions, deletions or substitutions. In the preferred embodiment theviral construct comprises sequences from a lentivirus genome, such asthe HIV genome or the SIV genome.

The viral construct preferably comprises sequences from the 5′ and 3′LTRs of a lentivirus. More preferably the viral construct comprises theR and U5 sequences from the 5′ LTR of a lentivirus and an inactivated orself-inactivating 3′ LTR from a lentivirus. The LTR sequences may be LTRsequences from any lentivirus from any species. For example, they may beLTR sequences from HIV, SIV, FIV or BIV. Preferably the LTR sequencesare HIV LTR sequences.

The viral construct preferably comprises an inactivated orself-inactivating 3′ LTR. The 3′ LTR may be made self-inactivating byany method known in the art. In the preferred embodiment the U3 elementof the 3′ LTR contains a deletion of its enhancer sequence, preferablythe TATA box, Sp1 and NF-kappa B sites. As a result of theself-inactivating 3′ LTR, the provirus that is integrated into the hostcell genome will comprise an inactivated 5′ LTR.

Optionally, the U3 sequence from the lentiviral 5′ LTR may be replacedwith a promoter sequence in the viral construct. This may increase thetiter of virus recovered from the packaging cell line. An enhancersequence may also be included. Any enhancer/promoter combination thatincreases expression of the viral RNA genome in the packaging cell linemay be used. In the preferred embodiment the CMV enhancer/promotersequence is used.

In one embodiment the viral construct comprises a gene that encodes aprotein that is desirably expressed in one or more cells of a transgenicanimal. Preferably the gene of interest is located between the 5′ LTRand 3′ LTR sequences. Further, the gene of interest is preferably in afunctional relationship with other genetic elements, for exampletranscription regulatory sequences such as promoters and/or enhancers,to regulate expression of the gene of interest in a particular manneronce the transgene is incorporated into the host genome. In certainembodiments, the useful transcriptional regulatory sequences are thosethat are highly regulated with respect to activity, both temporally andspatially.

Preferably the gene of interest is in a functional relationship withinternal promoter/enhancer regulatory sequences. An “internal”promoter/enhancer is one that is located between the 5′ LTR and the 3′LTR sequences in the viral construct and is operably linked to the genethat is desirably expressed.

The internal promoter/enhancer may be any promoter, enhancer orpromoter/enhancer combination known to increase expression of a genewith which it is in a functional relationship. A “functionalrelationship” and “operably linked” mean, without limitation, that thegene is in the correct location and orientation with respect to thepromoter and/or enhancer that expression of the gene will be affectedwhen the promoter and/or enhancer is contacted with the appropriatemolecules.

The internal promoter/enhancer is preferably selected based on thedesired expression pattern of the gene of interest and the specificproperties of known promoters/enhancers. Thus, the internal promoter maybe a constitutive promoter. Non-limiting examples of constitutivepromoters that may be used include the promoter for ubiquitin, CMV(Karasuyama et al J. Exp. Med. 169:13 (1989), β-actin (Gunning et al.Proc. Natl. Acad. Sci. USA 84:4831-4835 (1987) and pgk (see, forexample, Adra et al. Gene 60:65-74 (1987), Singer-Sam et al. Gene32:409-417 (1984) and Dobson et al. Nucleic Acids Res. 10:2635-2637(1982)). Alternatively, the promoter may be a tissue specific promoter.Several non-limiting examples of tissue specific promoters that may beused include lck (see, for example, Garvin et al. Mol. Cell Biol.8:3058-3064 (1988) and Takadera et al. Mol. Cell Biol. 9:2173-2180(1989)), myogenin (Yee et al. Genes and Development 7:1277-1289 (1993),and thy1 (Gundersen et al. Gene 113:207-214 (1992). In addition,promoters may be selected to allow for inducible expression of thetransgene. A number of systems for inducible expression using such apromoter are known in the art, including the tetracycline responsivesystem and the lac operator-repressor system. It is also contemplatedthat a combination of promoters may be used to obtain the desiredexpression of the gene of interest. The skilled artisan will be able toselect a promoter based on the desired expression pattern of the gene inthe resulting transgenic animal.

An internal enhancer may also be present in the viral construct toincrease expression of the gene of interest. For example the CMVenhancer (Karasuyama et al J. Exp. Med. 169:13 (1989) may be used incombination with the chicken β-actin promoter. Again, one of skill inthe art will be able to select the appropriate enhancer based on thedesired expression pattern.

The gene of interest is not limited in any way and includes any genethat the skilled practitioner desires to have integrated and/orexpressed in a transgenic animal. For example, the gene of interest maybe one that encodes a protein that modifies a physical characteristic ofthe transgenic animal, such as a protein that modifies size, growth, ortissue composition. In another example the gene of interest may encode aprotein of commercial value that may be harvested from the transgenicanimal.

In addition, more than one gene of interest may be placed in functionalrelationship with the internal promoter. For example a gene encoding amarker protein may be placed after the primary gene of interest to allowfor identification of cells that are expressing the desired protein. Inone embodiment a fluorescent marker protein, preferably greenfluorescent protein (GFP), is incorporated into the construct along withthe gene of interest. If a second reporter gene is included, an internalribosomal entry site (IRES) sequence is also preferably included. TheIRES sequence may facilitate the expression of the reporter gene

The viral construct may also contain additional genetic elements. Thetypes of elements that may be included in the construct are not limitedin any way and will be chosen by the skilled practitioner to achieve aparticular result. For example, a signal that facilitates nuclear entryof the viral genome in the target cell may be included. An example ofsuch a signal is the HIV-1 flap signal.

Further, elements may be included that facilitate the characterizationof the provirus integration site in the genome of the animal. Forexample, a tRNA amber suppressor sequence may be included in theconstruct.

In addition, the construct may contain one or more genetic elementsdesigned to enhance expression of the gene of interest. For example, awoodchuck hepatitis virus responsive element (WRE) may be placed intothe construct (Zufferey et al. J. Virol. 74:3668-3681 (1999); Deglon etal. Hum. Gene Ther. 11:179-190 (2000)).

A chicken β-globin insulator may also be included in the viralconstruct. This element has been shown to reduce the chance of silencingthe integrated provirus in the transgenic animal due to methylation andheterochromatinization effects. In addition, the insulator may shieldthe internal enhancer, promoter and exogenous gene from positive ornegative positional effects from surrounding DNA at the integration siteon the chromosome.

Any additional genetic elements are preferably inserted 3′ of the geneof interest.

In a specific embodiment, the viral vector comprises: a cytomegalovirus(CMV) enhancer/promoter sequence; the R and U5 sequences from the HIV 5′LTR; the HIV-1 flap signal; an internal enhancer; an internal promoter;a gene of interest; the woodchuck hepatitis virus responsive element; atRNA amber suppressor sequence; a U3 element with a deletion of itsenhancer sequence; the chicken β-globin insulator; and the R and U5sequences of the 3′ HIV LTR.

The viral construct is preferably cloned into a plasmid that may betransfected into a packaging cell line. The preferred plasmid preferablycomprises sequences useful for replication of the plasmid in bacteria.

Production of Virus

Any method known in the art may be used to produce infectious retroviralparticles whose genome comprises an RNA copy of the viral constructdescribed above.

Preferably, the viral construct is introduced into a packaging cellline. The packaging cell line provides the viral proteins that arerequired in trans for the packaging of the viral genomic RNA into viralparticles. The packaging cell line may be any cell line that is capableof expressing retroviral proteins. Preferred packaging cell linesinclude 293 (ATCC CCL X), HeLa (ATCC CCL 2), D17 (ATCC CCL 183), MDCK(ATCC CCL 34), BHK (ATCC CCL-10) and Cf2Th (ATCC CRL 1430). The mostpreferable cell line is the 293 cell line.

The packaging cell line may stably express the necessary viral proteins.Such a packaging cell line is described, for example, in U.S. Pat. No.6,218,181. Alternatively a packaging cell line may be transientlytransfected with plasmids comprising nucleic acid that encodes thenecessary viral proteins.

In one embodiment a packaging cell line that stably expresses the viralproteins required for packaging the RNA genome is transfected with aplasmid comprising the viral construct described above.

In another embodiment a packaging cell line that does not stably expressthe necessary viral proteins is co-transfected with two or more plasmidsessentially as described in Yee et al. (Methods Cell. Biol. 43A, 99-112(1994)). One of the plasmids comprises the viral construct comprisingthe transgene. The other plasmid(s) comprises nucleic acid encoding theproteins necessary to allow the cells to produce functional virus thatis able to infect the desired host cell.

The packaging cell line may not express envelope gene products. In thiscase the packaging cell line will package the viral genome intoparticles that lack an envelope protein. As the envelope protein isresponsible, in part, for the host range of the viral particles, theviruses are preferably pseudotyped. Thus the packaging cell line ispreferably transfected with a plasmid comprising sequences encoding amembrane-associated protein that will permit entry of the virus into ahost cell. One of skill in the art will be able to choose theappropriate pseudotype for the host cell that is to be used. Forexample, in one embodiment the viruses are pseudotyped with thevesicular stomatitis virus envelope glycoprotein (VSVg). In anotherembodiment, a mutant ecotropic envelope protein is used, such as theecotropic envelope protein 4.17 (Powell et al. Nature Biotechnology18(12):1279-1282 (2000)). In addition to conferring a specific hostrange the pseudotype may permit the virus to be concentrated to a veryhigh titer and may enhance safety by preventing the virus from infectingundesired cell types.

In the preferred embodiment a packaging cell line that does not stablyexpress viral proteins is transfected with the viral construct, a secondvector comprising the HIV-1 packaging vector with the env, nef, 5′LTR,3′LTR and vpu sequences deleted, and a third vector encoding an envelopeglycoprotein. Preferably the third vector encodes the VSVg envelopeglycoprotein.

The recombinant virus is then preferably purified from the packagingcells, titered and diluted to the desired concentration.

Transgenic Animals

In order to make transgenic animals, an oocyte or one or more embryoniccells are infected with the recombinant virus produced as describedabove. One of skill in the art will recognize that the method ofinfection and the treatment of the cell following infection will dependupon the type of animal from which the cell is obtained. For example,mammalian cells are preferably implanted in a pseudopregnant femalefollowing infection while for the generation of transgenic birds orfish, the virus is preferably delivered to a laid egg and thusimplantation is not required.

While early methods of making transgenic animals required the cells tobe rapidly dividing, there is no such requirement in the methods of thepresent invention. Thus the cell may be contacted at any point indevelopment. In the preferred embodiment, a zygote is contacted with therecombinant virus.

The cells to be infected with the virus may be obtained by any methodknown in the art and appropriate for the specific species in which it isdesired to make a transgenic animal. For example, the recovery offertilized mouse oocytes is described in Hogan et al. (Manipulating theMouse Embryo: A Laboratory Manual. 2^(nd) ed. Cold Spring HarborLaboratory Press, NY (1994)). A method for obtaining fertilized ratoocytes is described in Armstrong et al. (Biol. Reprod. 39, 511-518(1998)).

It is not necessary that the cells be contacted after fertilization. Inone embodiment, the virus is delivered to unfertilized ova. Developmentmay then be initialized, for example by in vitro fertilization.

Delivery of the Virus

The virus may be delivered to the cell in any way that allows the virusto infect the cell. Preferably the virus is allowed to contact the cellmembrane. Two preferred methods of delivering the virus to mammaliancells, injection and direct contact, are described below.

Injection

In a first embodiment the virus is injected into the perivitelline spacebetween the zona pellucida and the cell membrane of a single-cellzygote. Preferably less than 50 picoliters of viral suspension isinjected, more preferably less than 25 picoliters and even morepreferably about 10 picoliters.

The virus is preferably present in a viral suspension and may beinjected by any method known in the art. The viral suspension ispreferably injected through a hydraulic injector. More preferably aglass micropipette is used to inject the virus. In one embodiment amicropipette is prepared by pulling borosilicate glass capillary on apipette puller. The tip is preferably opened and beveled toapproximately 10 μm. The lentiviral suspension may be loaded into themicropipette from the tip using gentle negative pressure.

In one embodiment the cell is stabilized with a holding pipette mountedon a micromanipulator, such as by gentle negative pressure against afire-polished pipette, and a second micromanipulator is used to directthe tip of a micropipette into the space between the zona pellucida andthe cell membrane, where the virus is injected.

Direct Contact

In another embodiment the zona pellucida is removed from the cell toproduce a denuded embryo and the cell membrane is contacted with thevirus. The zona pellucida may be removed by any method known in the art.Preferably it is removed by enzymatic treatment. For example, treatmentwith pronase may be used to remove the zona pellucida while the cellmembrane is kept intact. Alternatively, the cell may be placed in mediaat pH at which the zona pellucida dissolves while the cell membraneremains intact. For example the cell may be incubated in an acidicTyrode's solution at room temperature for several minutes. Once the zonapellucida is removed, any method that allows for the virus to contactthe cell membrane may be used. Preferably, the cell is incubated in asolution containing the virus. Even more preferably, the solution ismedia that facilitates survival of the cell.

In this embodiment, the cells are preferably contacted with the virus inculture plates. The virus may be suspended in media and added to thewells of a multi-well culture plate. The cells may then be plated in theindividual wells. The media containing the virus may be added prior tothe plating of the cells or after the cells have been plated. Preferablyindividual cells are incubated in approximately 10 μl of media. However,any amount of media may be used as long as an appropriate concentrationof virus in the media is maintained such that infection of the host celloccurs.

The cells are preferably incubated with the virus for a sufficientamount of time to allow the virus to infect the cells. Preferably thecells are incubated with virus for at least 1 hour, more preferably atleast 5 hours and even more preferably at least 10 hours.

Both the injection and direct contact embodiments may advantageously bescaled up to allow high throughput transgenesis. Because of the relativesimplicity of the injection technique, it is possible to inject manyembryos rapidly. For example, it is possible to inject more than 200fertilized oocytes in less than one hour. With regard to the directcontact embodiment, any number of embryos may be incubated in the viralsuspension simultaneously. This may be accomplished, for example, byplating the desired number of single-cell zygotes in multi-well tissueculture plates containing the virus suspended in media appropriate forthe survival and growth of the cells.

In both embodiments, any concentration of virus that is sufficient toinfect the cell may be used. Preferably the concentration is at least 1pfu/μl, more preferably at least 10 pfu/μl, even more preferably atleast 400 pfu/μl and even more preferably at least 1×10⁴ pfu/μl.

Following infection with the virus, the cells are preferably implantedin an animal. More preferably cells infected with the virus areimplanted in pseudo-pregnant animals of the same species from which theinfected cells were obtained. Methods of creating pseudo-pregnancy inanimals and implanting embryos are well known in the art and aredescribed, for example, in Hogan et al. (Manipulating the Mouse Embryo:A Laboratory Manual. 2^(nd) ed. Cold Spring Harbor Laboratory Press, NY(1994)).

In the preferred embodiment early stage embryos (approximately 0-2.5days p.c.) still with an intact zona pellucida are transferred to theoviduct of timed pseudopregnant female (preferably 0.5 days p.c.), whileembryos that have reached the blastocyst stage are transferred to theuterus of timed pseudopregnant females (preferably 2.5 days p.c.).Denuded embryos are preferably cultured in vitro until they reach themorula or blastocyst stage (48 to 72 hours in culture), and are thenimplanted into appropriately timed pseudopregnant females.

The embryos and resulting animals may be analyzed, for example forintegration of the transgene, the number of copies of the transgene thatintegrated, the location of the integration, the ability to transmit thetransgene to progeny and expression of the transgene. Such analysis maybe carried out at any time and may be carried out by any methods knownin the art. Standard techniques are described, for example, in Hogan etal. (supra).

The methods of infecting cells disclosed above do not depend uponspecies-specific characteristics of the cells. As a result, they arereadily extended to all mammalian species.

Initial experiments with mice indicate that of those animals thatdevelop to full term, 80-90% carried at least one copy of the transgeneand that, of these, approximately 85% express the gene of interest. Ofthe transgenic animals about 25% carry only 1 or 2 copies of thetransgene. The highest number of proviral insertions observed was about30. Of the animals that carried only 1 or 2 copies of the transgene,about 80% expressed the gene of interest.

As discussed above, the modified retrovirus can be pseudotyped to conferupon it a broad host range. One of skill in the art would also be awareof appropriate internal promoters to achieve the desired expression of agene of interest in a particular animal species. Thus, one of skill inthe art will be able to modify the method of infecting cells to createtransgenic animals of any species.

For example, transgenic birds are created by delivering a modifiedretrovirus, as described above, to the primordial germ cells of earlystage avian embryos. In one embodiment, freshly laid eggs are obtainedand placed in a temperature controlled, humidified incubator.Preferably, the embryonic blastodisc in the egg is gradually rotated tolie on top of the yolk. This may be accomplished by any method known inthe art, such as by gently rocking the egg regularly, preferably every15 minutes. Approximately 36 hours later, the modified retrovirus isdelivered into the space between the embryonic disk and theperivitelline membrane. Preferably about 50 nL of viral solution isdelivered, more preferably about 100 nL of viral solution is delivered,and even more preferably about 200 nL of viral solution is delivered.The viral solution may be delivered by any method known in the art fordelivering compositions to the inside of an egg. In the preferredembodiment a window is opened in the shell, the viral solution isinjected through the window and the shell window is closed. The eggs arepreferably incubated until hatching. The eggs will hatch afterapproximately 20 days, depending upon the particular avian species fromwhich they are obtained. Hatched chicks are preferably raised to sexualmaturity and mated. The transgenic offspring of the founder animals maybe identified by any method known in the art, such as Southern blot, PCRand expression analysis.

In another embodiment, transgenic fish are created by delivering themodified retrovirus, described above, to single-cell fish embryos.Fertilized fish eggs are collected by any method known in the art. Themodified retrovirus is then preferably delivered to the space betweenthe chorion and the cell membrane. This may be accomplished, forexample, by loading the modified retrovirus in solution into a glasspipette. The pipette may then be used to pierce the chorion membrane anddeliver the viral suspension. Preferably about 50 nL of viral solutionis delivered, more preferably about 100 nL of viral solution isdelivered, and even more preferably about 200 nL of viral solution isdelivered. Injected embryos are preferably returned to atemperature-controlled water tank and allowed to mature. At sexualmaturity the founder fish are preferably mated and their progenyanalyzed for the presence of the transgene by any method known in theart.

As mentioned above, the methods of the present invention will also proveuseful in techniques for identifying genes that are involved in specificbiological processes, such as gene trap assays and large-scalemutagenesis screens.

Gene trap experiments allow the identification and cloning of a genethat is expressed in a particular tissue or cell type, and/or at aparticular time, based solely on its pattern of expression. Genetrapping relies on the capture of the splicing donor of an mRNA byectopically inserting a downstream splice acceptor, in this case,carried within an integrated provirus. Gene trapping has beensuccessfully used in several model systems, including the fruit flyDrosophila, mammalian cells in culture, and mouse ES cells (which havethe advantage of being able to be used to derive mice afterwards forfurther analysis). Gene trapping in cell culture has the advantage ofbeing fast and inexpensive, but is limited by the inability of the cellsto differentiate into specific cell types. Thus, gene trappingexperiments in mammalian cell lines in culture usually yield onlyhousekeeping genes expressed non-specifically in any mammalian cell, orcell-specific genes that are only expressed by the particular cell linein vitro. Because cell lines often show incomplete degrees ofdifferentiation, the complement of tissue-specific genes expressed bythese cells is limited. Furthermore, there are many tissues for whichrepresentative cell lines do not exist.

The use of the above-described recombinant lentiviral vectors for thepurposes of gene trapping is facilitated by the self-inactivatingmutation in the U3 enhancer element of the 3′ LTR. The lack oftranscriptional activity from the integrated 5′ LTR ensures that anytranscription of a reporter element in the provirus is driven byupstream regulatory sequences to the insertion that have been “trapped,”rather than from the viral promoter itself.

Thus, one embodiment of the present invention concerns a method ofidentifying genes that are expressed in a particular tissue and/or at aparticular time during the development of an organism. Aself-inactivating viral construct is made that preferably comprises asplice acceptor sequence and a sequence encoding a reporter gene.Modified retroviral particles are made using the viral construct asdescribed above and used to infect embryonic cells. Tissues from thefounder animal or its progeny are analyzed for the presence of thereporter to determine the temporal and/or spatial pattern of expression.Messenger RNA is collected from the tissues of the animals that expressthe reporter protein in the time and place of interest. The “trapped”gene may then be identified by any method known in the art. Preferably,oligonucleotides that are complementary to the reporter gene may thenused in a reverse transcription reaction to produce a cDNA that containsthe sequences of the trapped gene that flank the provirus. The cDNA maythen be cloned into a plasmid from which the gene may be identified bynucleotide sequencing.

Gene trap experiments are well known in the art and the skilled artisanwould be able to choose the reporter gene, splice acceptor sequence andany other genetic elements that would be useful to include in the viralconstruct based on the specific analysis that they have undertaken. Inaddition, by modifying the viral constructs, the technique can be usedto trap promoter or enhancer sequences. For promoter trap experiments,the reporter gene lacks any transcriptional regulatory elements, and isonly expressed when the virus integrates next to an active promoter. Forthe enhancer trap, the reporter gene is positioned downstream of aminimal promoter that lacks transcriptional activity, and is onlyexpressed when the virus integrates next to an active enhancer.

Another important paradigm by which biologists study gene function is todisrupt the function of an endogenous gene and, from the mutantphenotype that results, deduce the normal role of that gene in theorganism. One way of isolating genes that are important in a particularbiological process under study is to perform large-scale mutagenesis togenerate animals that are phenotypically mutant in that process and thento isolate the gene that is disrupted in the mutant animal and that isthus responsible for the mutant phenotype. In most such experiments,either radiation or chemicals have been used to induce deletions ornonsense mutations. However, the genes carrying mutations induced byradiation or chemicals are difficult to isolate because no handle isavailable with which to clone the gene. Rather, these mutations must beidentified by positional cloning, a slow and painstaking process inwhich the mutation of interest is systematically mapped relative toknown genetic markers in the genome, with the goal of graduallynarrowing down and pinpointing the locus of the mutated gene.

In contrast, a powerful technique that has been used successfully in thefruit fly Drosophila melanogaster is that of insertional mutagenesis, inwhich genes are disrupted when an exogenous piece of DNA is insertedwithin the coding sequence of the gene. The great advantage ofinsertional mutagenesis is that, because the sequence of the exogenousdisrupting DNA is known, one can directly clone out that piece of DNAand the flanking sequence that corresponds to the gene of interest thathas been disrupted. Thus, in contrast to traditional positional cloningstrategies used in chemical mutagenesis which may take up to 3 yearsafter the isolation of the mutant, identification and characterizationof the mutated gene of interest in an insertional mutagenesis strategyis reduced to just a week or so. The main limitation to the applicationof insertional mutagenesis in organisms other than the fruit fly is thelack of a DNA element, such as the transposon used in Drosophila, thatis able to stably integrate and mark its position in the genome at thegermline or one-cell embryo stage.

The lentiviral vectors described above can be effective tools forlarge-scale mutagenesis to identify genes involved in specificbiological processes. The modified lentiviruses of the present inventionare easily delivered to the germline, and pseudotyping of the viruseswith an envelope glycoprotein, such as VSVg, allows the concentration ofthe virus to extremely high titers. Thus in one embodiment mutagenesisis achieved by delivery of the modified retrovirus to the cell membraneof embryonic cells.

The ability of transgenic animals made by the methods of the presentinvention to express a gene of interest at high levels suggests that theintegrated proviruses are not silenced by methylation. Previousmutagenesis screens using MoLV-based retroviruses have been limited bythe observation that, in addition to the provirus, flanking genomicsequences are frequently found to be methylated and inactivated. Thismethylation complicates the analysis because it becomes difficult todistinguish whether the mutant phenotype is due to the disruption of thegene into which the provirus has inserted, or due to the inactivation ofany one of several surrounding genes by methylation.

By delivering modified lentiviruses to embryos according to the methodsof the present invention, insertional mutagenesis strategies can beapplied to any animal species, including model genetic organisms such asXenopus, zebrafish, mouse, rat, and zebra finch. Early-stage embryos,consisting of several cells, will preferably be targeted because theresulting mosaicism increases the number of unique mutagenic events thatcan be screened.

The modified lentiviruses integrate randomly into the genome of thetarget zygotes, including that of the germ cells. Thus, some proportionwill disrupt coding sequences. The embryos are preferably raised tosexual maturity, mated, and the progeny are screened for mutantphenotypes of interest. Once a mutant is identified, selective breedingusing standard methods is preferably used to isolate the particularinsertion(s) responsible for the phenotype.

Once a mutant line is established, the mutated locus is preferablyidentified using the provirus as a handle for cloning. In oneembodiment, an origin of replication and antibiotic resistance gene isincluded in the viral construct. In this embodiment, genomic DNA fromthe mutant is preferably isolated, randomly cleaved with an appropriaterestriction enzyme, and the linear fragments circularized by ligation.The ligation mixture is then transformed into bacterial cells and platedon antibiotic plates. The plasmid DNA from any colonies that arise isisolated and preferably used as a template for inverse PCR withoppositely oriented, adjacent primers complementary to sequences in theprovirus. The amplified DNA molecule(s) is then sequenced to acquire theflanking regions to the integration site, corresponding to the gene(s)mutated.

In another embodiment, inclusion of the tRNA amber suppressor sequencein the provirus allows for the rapid generation of genomic librariescontaining the flanking regions of the integration loci, representingthe disrupted gene(s). Once these flanking regions are sequenced, theycan be compared against the genomic sequence database for that animal todetermine candidate gene(s) of interest.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and fall within the scope of theappended claims.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

Example 1

Transgenic mice were generated that expressed a heterologous protein,green fluorescent protein (GFP). GFP expression was controlled bymanipulating the genetic elements in the viral construct employed tocreate the transgenic mice. For example, a viral construct, FUGW,comprising a ubiquitous promoter was used to produce transgenic micethat expressed GFP in every cell. Inclusion of a nuclear localizationsignal produced transgenic mice that had GFP localized in the nucleus oftheir cells. A viral construct with a lymphocyte specific promoterproduced mice that expressed GFP in lymphocytes, while a viral constructwith a muscle specific promoter produced mice that specificallyexpressed GFP in muscle cells.

A. Viral Constructs

A viral construct according to the present invention was created usingthe HR′CS-G plasmid (Miyoshi H, Blomer U, Takahashi M, Gage F H, Verma IM. J Virol. 72(10):8150-7 (1998)). This plasmid is based on the HIV-1HXB2 proviral DNA (see U.S. Pat. No. 6,013,516).

1. Generation of a Vector Expressing GFP from a Ubiquitous Promoter,FUGW

The HIV-1 flap sequence (SEQ ID NO: 1) was inserted into the HR′CS-Gvector. A 147 base pair sequence containing the flap region (Zennou, V.,Petit, C., Guetard, D., Nerhbass, U., Montagnier, L., Charneau, P. Cell.101(2), 173-185 (2000)) was PCR amplified from a plasmid encoding thegenome of HIV NLA4.3. The 5′ PCR primer encoded BglII and PacI sites.The 3′ PCR primer contained the BamHI site. The resulting PCR productwas digested with BglII and BamHI enzymes and inserted into the BamHIsite of the HR′CS-G vector. The resulting plasmid was called Hflap,representing the Hflap sequence that the plasmid contains.

The ubiquitin promoter (SEQ ID NO: 2) was then inserted into the Hflapplasmid. The 1.2 Kb sequence encoding the human polyubiquitin C promoterwas excised with BglII and BamHI enzymes and inserted into the BamHIsite of Hflap. The resulting plasmid was called HflapUbi, representingthe HflapUbi sequence that the plasmid contains.

A multi-cloning site was then inserted into HflapUbi. Twooligonucleotides were designed that encoded the following restrictionsites: BamHI HpaI XhoI AscI EcoRI BglII. The oligos were hybridized andinserted into the BamHI site of HflapUbi. The resulting plasmid wascalled FUMCS.

A nucleic acid sequence encoding GFP (SEQ ID NO: 3) was then insertedinto HflapUbi. The 700 base pair sequence of GFP was digested with BamHIand XhoI and inserted into the XhoI site of HflapUbi, generatingHflapUbiG which represents the HflapUbiG sequence that the plasmidcontains. The resulting plasmid was called FUG.

The woodchuck hepatitis virus regulator element (WRE; SEQ ID NO: 4) wasthen inserted into HflapUbiG. The 500 bp sequence of WRE (Zufferey, R.,Donello, J. E., Trono, D., Hope T. J. (1999). J. Virol. 73(4), 2886-92)was excised with SalI and XhoI and inserted into the XhoI site ofHflapUbiG, generating a plasmid containing the HflapUbiGWRE sequence(SEQ ID NO: 8). The resulting plasmid was called FUGW (SEQ ID NO: 9). Amap of the FUGW viral vector is presented in FIG. 1A.

2. Generation of a Vector Expressing Nuclear-Localized GFP from aUbiquituous Promoter, FUH2BGW

In order to get specific nuclear localization of GFP, the histone 2B-GFPfusion sequence H2BGFP was cloned into FUGW. The histone 2B-GFP sequence(SEQ ID NO: 5) was digested with SalI and NotI. Both sites were bluntedwith T4 DNA polymerase and inserted into the HpaI site of FUMCS. Theresulting plasmid was called FUH2BGW.

3. Generation of a Vector Expressing GFP from a Lymphocyte-SpecificPromoter, FlckGW

To achieve lymphocyte specific GFP expression the murine lck promoter(SEQ ID NO: 6) was cloned into the FUGW vector. The ubiquitin promoterfrom FUGW was removed by excising with PacI and BamHI. The PacI site wasblunted using T4 DNA polymerase and the lck promoter was excised withSpeI and BamHI. The SpeI site was blunted using T4 DNA polymerase. Thelck promoter was the inserted into the PacI and BamHI sites of FUGW. Theresulting plasmid was called FlckGW.

4. Generation of a Vector Expressing GFP from a Muscle-SpecificPromoter, FmyoH2BGW

To achieve specific expression of GFP in the muscle of transgenic micethe myogenin promoter (SEQ ID NO: 7) was cloned into the FUGW construct.The mouse myogenin promoter was PCR amplified from a mouse genomic BAC.The 5′ PCR primer encoded a PacI site, and the 3′ PCR primer containedan XbaI site. The PCR product was digested with PacI and XbaI. Theubiquitin promoter was removed from the FUH2BGW vector by cutting withPacI and XbaI. The PacI-BamHI digested PCR product encoding the myogeninpromoter was inserted into the PacI and XbaI sites of the FUH2BGWvector.

The constructs described above were then used to prepare recombinantlentivirus. Briefly, replication-incompetent viral vectors, based on thehuman immunodeficiency virus-1 (HIV-1), were pseudotyped with thevesicular stomatitis virus envelope glycoprotein (VSVg), permitting thevirus to be concentrated to very high titers and conferring upon thevirus a broad host range. Pseudotyped lentiviruses were producedessentially as described in detail in Yee, J. K., Friedmann, T. & Burns,J. C. (1994). Methods Cell Biol. 43, 99-112; Burns, J. C., Friedmann,T., Driever, W., Burrascano, M., and Yee, J. K. (1993). Proc. Natl.Acad. Sci. USA. 90, 8033-8037; and Yee, J. K., Miyanohara, A., LaPorte,P., Bouic, K., Burns, J. C., and Friedmann, T. (1994). Proc. Natl. Acad.Sci. USA. 91, 9564-9568. Briefly, human fibroblasts 293 cells weretransfected with calcium phosphate/DNA coprecipitates of the followingplasmids, as described in Gorman, C., Padmanabhan, R. and Howard, B. H.(1983). Science. 221, 551-553:

The viral transfer vector described above with self-inactivating LTR;

the HIV-1 packaging vector Δ8.9 (Zufferey, R., Nagy, D., Mandel, R. J.,Naldini, L., and Trono, D. (1997). Nat. Biotechnol. 15(9), 871-875;Naldini, L., Blomer, U., Gallay, P., Ory, D., Mulligan, R., Gage, F. H.,Verman, I. M., and Trono, D. (1996). Science. 272(5259), 263-67) withenv, vpr, vpu, vif, nef, 5′LTR, 3′LTR, and ψ sequences deleted; and theVSVg envelope glycoprotein.

Viral supernatant was harvested 60 hours post-transfection, subjected tolow-speed centrifugation to remove cell debris, filtered through a 0.45μm nitrocellulose membrane, spun at 25,000 rpm for 1.5 hours toconcentrate, and resuspended in a small volume (one hundredth to onethousandth of the original volume) of phosphate-buffered saline (PBS),pH 7.4. The titer of the viral concentrate was approximately 1×10⁶pfu/μl as determined in 293 human fibroblasts measured by the number ofGFP-positive cells. The viral suspension was stored frozen at −80° C.

B. Production of Transgenic Mice and Rats

The lentivirus was used to produce transgenic mice and rats.

1. Superovulation and Embryo Collection

Female mice and rats were superovulated with a combination of pregnantmare's serum (PMS) and human chorionic gonadotropin (hCG) as describedin Hogan, B., Beddington, R., Costantini, F., and Lacy, E. (1994).Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring HarborLaboratory Press. “Superovulation” refers to administering gonadotropinsto female mammals prior to mating to increase the number of eggs thatare ovulated. Prepubescent female mice (approximately 25 days of age andweighing between 12.5 and 14 grams) were injected intraperitoneally with5 IU of PMS (Sigma G 4527, 25 IU/ml in 0.9% NaCl) between 1 and 3 p.m.on day −2, followed by 5 IU of HCG (Sigma C 8554, 25 IU/ml in 0.9% NaCl)48 hours later on day 0. Prepubescent female rats between 28-30 days ofage and weighing between 70 and 80 grams were injected intraperitonallywith 25 IU of PMS between 1 and 3 p.m. on day −2, followed by 5 IU ofHCG 48 hours later on day 0. For both rats and mice, hormone-treatedfemales were caged overnight with fertile males (2-3 months of age) tomate. On the morning of day 1, females were checked for copulationplugs.

Female mice were sacrificed for embryo collection around 10 a.m. on themorning of day 1, while female rats were sacrificed for collectionaround 1 p.m. on the afternoon of day 1. Embryos were collected frommice and rats essentially following the procedure described in Hogan,B., Beddington, R., Costantini, F., and Lacy, E. (1994) Manipulating theMouse Embryo: A Laboratory Manual. Cold Spring Harbor Laboratory Press.Briefly, animals were sacrificed by CO₂ inhalation, and the oviductswere excised and transferred to a dish containing M2 medium at roomtemperature. Newly fertilized embryos, enclosed by cumulus mass cells,were released from the swollen ampullae (the upper portion of theoviduct) by gently tugging and opening the walls of the ampullae withfine forceps. The embryos were then transferred to a dish containing ahyaluronidase solution (Sigma H 3884, 300 μg/ml in M2 medium), whichenzymatically digested the cumulus cells, thus releasing the embryos.When the cumulus cells were shed, the embryos were transferred to freshM2 medium to wash off the hyaluronidase solution and preserve theviability of the embryos. In rats, the cumulus cells were found toadhere tenaciously to the surface of some embryos and were difficult toremove completely. Thus in some cases the subsequent experimentalmanipulations with the zygotes were carried out with some of the cumuluscells still adhering. This did not seem to affect the outcome. Theembryos were then transferred to microdrops of M16 medium under mineraloil and cultured in a humidified 37° C. incubator under 5% CO₂ untilneeded.

2. Delivery of Lentiviruses to Single-Cell Embryos

Lentiviruses were delivered to the fertilized oocytes on the same day ofcollection, targeting only single-cell zygotes to minimize mosaicism.Infection with lentivirus derived from the FUGW viral construct willlead to integration of the provirus locus diagrammed in FIG. 1B. Twodifferent methods were used to deliver the lentiviruses to the embryos:

a. Microinjection of Lentiviruses into the Perivitelline Space ofSingle-Cell Embryos

Micropipettes were prepared by pulling borosilicate glass capillaries (1mm O.D., 0.7 mm I.D.) on a Sutter Instruments pipette puller. The tipwas cut at an angle to approximately 10 μm with a razor blade. Themicropipette was then inserted into the pipette holder of a CellTramhydraulic injector (Eppendorf). The lentiviral concentrate preparedabove was pipetted up and down to release any large aggregates ofcellular debris. The virus was centrifuged at low speed in a tabletopmicrofuge (1000 rpm for 1 min.), and removed from the top. The viralsuspension was then loaded into the micropipette from the tip usinggentle negative pressure from the CellTram.

One-cell embryos were transferred to a microdrop of M2 medium on a slideand covered with mineral oil to maintain the osmolarity. The slide wasmounted on the stage of an inverted light microscope, and the injectionprocedure was monitored under 400× magnification. Embryos were held inplace against a fire-polished pipette using gentle negative pressure.The pipette holder with the virus was loaded onto a micromanipulator(Leitz). Using the micromanipulator to guide the pipette, the tip waspushed through the zona pellucida into the region between the zonapellucida and the oocyte cell membrane. Using gentle positive pressure,approximately 10 nanoliters of the viral concentrate was delivered intothe perivitelline space. The micropipette was then withdrawn from thezygote. After the injection, the embryos were sorted and those that werelysed, abnormal, or at the 2-cell stage were discarded. The remainingembryos were transferred to M16 microdrops under oil and cultured in a37° C. incubator under 5% CO₂ until implantation.

b. Co-Incubation of Denuded Single-Cell Embryos with Lentiviruses

The zona pellucida of the fertilized oocytes was removed by incubationin either an acidic Tyrode's solution (Hogan, B., Beddington, R.,Costantini, F., and Lacy, E. (1994). Manipulating the Mouse Embryo: ALaboratory Manual. Cold Spring Harbor Laboratory Press) or a 0.5%pronase solution in M2 medium at 37° C. in a humidified 5% Co₂ incubatorfor several minutes. When the zonae appeared to be dissolved, embryoswere washed in excess M2 medium and then transferred into 10 μlmicrodrops of viral suspension under mineral oil. Embryos were culturedindividually in separate microdrops to prevent them from adhering to oneanother. The viral suspension was diluted to various concentrations toroughly control the average number of proviral integrations expected pertransgenic genome. Virus in the microdrops was diluted to 2×10⁴ pfu/μl,400 pfu/μl, and 8 pfu/μl. Zygotes were incubated in the viral suspensionfor at least 4-6 hours before implantation to allow viral entry into thecell.

3. Transfer of Embryos into Recipient Females

Timed pseudopregnant females to host the treated embryos were preparedby mating sexually mature females in estrus to vasectomized, maturemales the night before the intended day of implantation. Appropriatefemales were selected from a colony of 30-40 females by taking vaginalsmears and examining them for the cell types typical of the estrusphase. Males were vasectomized by tying off the vas deferens at twoseparate locations, approximately 5-6 mm apart, then cauterizing theintervening segment to sever the tube. Males were vasectomized at least2 weeks prior to the mating to ensure that all remaining sperm in thegenital tract were dead at the time of mating.

Embryos infected with lentivirus were transferred into host females assoon as possible to achieve maximum rates of implantation. Early-stageembryos (0-2.5 days p.c.) with an intact zona pellucida were transferredto the oviduct of timed pseudopregnant females (0.5 days p.c.), whileembryos that had reached the morula or blastocyst stage were transferredto the uterus of timed pseudopregnant females (2.5 days p.c.). Ingeneral, no more than 30 embryos were transferred bilaterally into theuterus. These procedures were carried out essentially as described in(Hogan, B., Beddington, R., Costantini, F., and Lacy, E. (1994).Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring HarborLaboratory Press). Pregnancy and delivery of the transgenic litter wasas usual.

C. Analysis of Transgenic Animals

Animals in the resulting litters were analyzed for the presence of thetransgene and the number of insertions of the transgene by standardSouthern blot analysis (Sambrook, J., Fritsch, E. F., Maniatis, T.(1989). Molecular Cloning: A Laboratory Manual. Cold Spring Harborlaboratory Press.), cutting with PstI or BamHI and hybridizing against aGFP+WRE probe. For constitutive promoters, expression of GFP wasdetermined by directly viewing the skin of the animals under aconventional epifluorescence microscope. Some transgenic animals thatwere scored as negative for expression were actually expressing thetransgene at levels below that of detection by visual inspection with afluorescent microscope. In such cases, western blot analysis revealedthat animals in which GFP fluorescence was not detected by visualinspection did express the GFP protein in some tissues. Similarly,immunocytochemistry proved to be a more sensitive assay for determiningexpression. For the tissue-specific promoters, some proportion of thetransgenic litter was sacrificed during development at embryonic stages,and the translucent embryos were checked for spatially regulated GFPexpression under a fluorescent microscope. Expression results wereconfirmed by histology. To test the ability of the founder animals totransmit the transgene to their progeny, animals positive by Southernanalysis were outcrossed to wild-type animals, and their progeny scoredfor transgenesis and expression as described above.

D. Results

In one set of experiments one-cell mouse and rat embryos were injectedin the perivitelline space with recombinant lentivirus as describedabove. In the first experiment, 17 founder mice developed to term from78 implanted embryos. Of these, 11 of the 17 founders expressed thetransgene as determined by directly viewing the animals under anepifluoresence microscope. Further, 11 of 15 (two mice died prior toanalysis), or approximately 73%, were found to carry the transgene bySouthern blot analysis. The average number of insertions in thetransgenic mice was 6.1. Several of the animals carried as few as 2insertions. These results are presented in FIG. 2.

In a second experiment, 56 founder mice developed to term from 119implanted embryos. Of these 45, or about 80%, were found to express thetransgene. Thus, in the two experiments 58 out of 73 founder mice,approximately 79.5%, carried the transgene. FIG. 3 shows the Southernblot analysis of proviral transgene insertions in these founder mice.

All GFP-positive mice carried an integrated provirus, and all animalswith two or more copies of the provirus expressed the transgene atlevels detectable by direct viewing of GFP fluorescence. The intensityof GFP fluorescence correlated positively with copy number, as estimatedqualitatively. All major tissues and organs, including skin, bone,skeletal muscle, cardiac muscle, lung, liver, thymus, spleen, stomach,intestine, kidney, brain, retina and gonads were GFP positive.

In a third experiment, five rats developed to term from embryos injectedwith lentivirus created from the FUGW construct. Two of the five ratswere found to express the transgene as determined by brightfield andfluorescent images of the paws of the newborn rats (FIG. 4A). Pup R4expresses GFP in the paw, as well as in all other tissues and organsthat were examined (FIG. 4A). FIG. 4B shows the Southern blot analysisof the proviral insertions in these founder rats and indicates that pupR4 carries 4 copies of the proviral insert.

In a continuation of this experiment, out of 22 founder rat pups bornfrom 130 implanted embryos, 13 (59.1%) carried one or more proviralinsertions as determined by Southern blot analysis and 9 (40.9%)expressed GFP at levels detectable by directly viewing the skin under afluorescent microscope. GFP positive founders were crossed to wild-typeanimals, and F1 progeny rats carrying as few as one copy of the provirusexpressed GFP, as determined by direct viewing with a fluorescentmicroscope, indicating that the GFP-expressing transgene is not silencedby transmission through the germline.

In another set of experiments denuded mouse embryos were incubated indecreasing concentrations of recombinant lentivirus. A rough correlationwas seen between the titer of virus in which embryos were incubated andthe number of proviral insertions. At a 1:50 dilution from a stock of1×10⁶ pfu/μl 5 founder mice that reached term (from 29 implantedembryos) were found to be transgenic. All of these animals carried atleast 6 proviral insertions. The average number of insertions was 7.2.Of these, 4, or 80%, were found to express the transgene. At a 1:250dilution five out of 7 founder mice that reached, term (from 18implanted embryos) were found to be transgenic and express thetransgene. In these mice the average number of insertions was 3.8, withtwo of the animals carrying only one or two copies of the transgene.Finally, at a dilution of 1:1250 only one of the 8 founders (from 40implanted embryos) was found to be transgenic, with a single insertion.This founder also expressed the transgene. FIG. 5 shows the Southernblot analysis of proviral transgene insertions in these founder mice.FIG. 6 shows GFP expression in one of the founder mice. A second trialwith a 1:250 dilution gave comparable results. Eight of 11 founder mice(from 59 implanted embryos) were transgenic, with seven expressing thetransgene. The transgenic mice had an average of 2.6 insertions.

Following outcrossing to wild-type animals, progeny were analyzed forproviral transgene insertions by Southern blot (FIG. 7) and for GFPexpression by viewing under an epifluoresence microscope (FIG. 8). Ascan be seen in Table 1, founder mice were able to transmit the transgeneto their progeny. In Table 1, “PV” represents founder transgenicsgenerated by injection of the lentivirus into the perivitelline space ofone-cell embryos while “Co-inc” represents founder transgenics generatedby co-incubation of the denuded embryos with lentivirus.

TABLE 1 No. insertions in No. Founder founder No. progeny expressingPV.13 6 7 6/7 PV.2 2 4 2/4 PV.10 2 7 1/7 PV.17 10 10 10/10 Co-inc.18 1212  9/12 Co-inc.2 0 9 0/9

Ubiquitous GFP expression similar to that of the founder animals wasobserved in transgenic F1 progeny, indicating that the provirus was notinactivated through one round of gametogenesis and development. Allanimals carrying two or more insertions of the FUGW provirus expressedGFP at levels detectable by direct fluorescence. However, amongtransgenic lines carrying one proviral insertion, approximately halfexpressed the transgene at levels detectable by direct fluorescence. Inone single insertion line in which GFP expression was not observed bydirect viewing, GFP was detectable by Western blot analysis in sometissues (brain, testes) but not in others (heart, lung, liver, kidney,spleen).

In a further experiment, single-cell mouse zygotes were injected in theperivitelline space with recombinant lentivirus derived from the FMH2BGWviral construct described above. This construct comprises ahistone2B-GFP fusion gene under the control of the myogenin promoter.The histone2B-GFP reporter was used to concentrate the fluorescence inthe nuclei, making the signal more intense. Zygotes were implanted inpseudopregnant female mice and then recovered from the uterus atembryonic day 11.5. As can be seen in FIG. 9, all six founder-mice weretransgenic as determined by Southern blot analysis of proviral transgeneinsertions. Of these, two animals were positive for tissue-specific GFPexpression at embryonic day 11.5 (FIG. 9).

FIG. 10 shows that at embryonic day 11.5, GFP expression is localized tothe somites and can be seen in the emerging muscles in the limb buds,eye and jaw. This expression pattern is consistent with myogeninexpression at this stage of development. FIG. 11 shows the results ofimmunofluorescence studies of sections of an embryonic day 11.5 embryocarrying the myogenin promoter driving GFP. Specific staining of somitetissues can be seen (FIG. 11). FIGS. 12 and 13 show furtherimmunofluorescence studies of cross-sections of an E11.5-embryo carryinga myogenin promoter driving GFP.

Fifteen-day old animals, derived from FMH2BGW-infected zygotes showedGFP fluorescence in the nuclei of skeletal muscle in the tongue, limbs,chest and jaw, but not in cardiac or smooth muscle or other non-muscletissues examined, reflecting the known specificity of myogeninexpression. F1 progeny from three independent founders expressedhistone2B-GFP exclusively in the skeletal muscle lineage. Furthermore,progeny carrying as few as one FMH2BGW proviral insertion expressedhistone2B-GFP in the appropriate tissue types at high levels detectableby direct viewing with a fluorescent microscope.

In a further experiment, a viral vector containing GFP driven by theT-lymphocyte promoter lck, FlckGW was delivered to the perivitellinespace by injection as described above. The resulting transgenic miceexpressed GFP exclusively in the thymus.

Example 2

Transgenic birds, such as chicken or quail, may be made by the methodsof the present invention.

Freshly laid chicken eggs (day 0) are placed in atemperature-controlled, humidified incubator at 38° C. The embryonicblastodisc is gradually rotated to lie on top of the yolk by gentlyrocking the eggs in the incubator every 15 minutes. A window is openedin the shell and the blastodisc is visualized in freshly laid eggs (0hours post-laying) or stage X embryos (36 hours post-laying).VSV-pseudotyped lentiviral particles in solution are loaded into a glasscapillary micropipette. To maximize the chances of targeting primordialgerm cells, virus is injected in the anterior regions of the 0 hourembryos and in the gonadal anlage of the 36 hours embryos. Approximately200 nL of viral solution are delivered into the space between theperivitelline membrane and the embryonic disk with the aid of ahydraulic injector. The shell window is then closed with a porous tapeto allow gas exchange between the embryo and the incubator atmosphere.The embryos are then incubated without rocking. The eggs will hatchafter approximately 20 days of incubation time. Hatched chicks areraised to sexual maturity and then mated. The eggs laid by the matedfemales are raised to hatching and the resulting transgenic chicks areidentified, such as by Southern blot, PCR or expression analysis.

Example 3

Transgenic zebra finch were made by the methods of the presentinvention.

Freshly laid zebra finch eggs (day 0) were placed in atemperature-controlled, humidified incubator at 38° C. The embryonicblastodisc was gradually rotated to lie on top of the yolk by gentlyrocking the eggs in the incubator every 15 minutes. A window was openedin the shell and the blastodisc was visualized. VSV-pseudotypedlentiviral particles in solution were loaded into a glass capillarymicropipette. The lentivirus was derived from the FUH2BGW viralconstruct described above. To maximize the chances of targetingprimordial germ cells, virus was injected in the anterior regions of 0hour embryos and in the gonadal anlage of 36 hour embryos. Approximately200 nL of viral solution are delivered into the space between theperivitelline membrane and the embryonic disk with the aid of ahydraulic injector.

The shell window was closed with a porous tape to allow gas exchangebetween the embryo and the incubator atmosphere. The embryos were thenincubated without rocking. FIG. 15 shows H2B-GFP expression in theextraembryonic tissue. FIG. 16 shows H2B-GFP expression inside of thezebra finch embryo, indicating that primordial germ cells carried andexpressed the transgene.

The eggs will hatch after approximately 20 days of incubation time.Hatched chimeric chicks are raised to sexual maturity and then mated.The eggs laid by the mated females are raised to hatching and theresulting transgenic chicks are identified, such as by Southern blot,PCR or expression analysis.

Example 4

Transgenic fish may be made by the methods of the present invention.Breeding pairs of fish are placed in a water tank with a grooved bottom,where fertilized eggs are deposited. Fertilized eggs (zygotes) arecollected and held in embryo medium on ice. Zygotes are aligned ingrooves formed in a slab of agarose. A modified lentivirus, as describedabove, is loaded into a glass capillary micropipette. The chorionmembrane surrounding the zygote is pierced with the glass micropipetteand 200 nL of viral solution are delivered into the space between thezygotic membrane and the chorion. Injected zygotes are returned to atemperature-controlled water tank and allowed to mature. At sexualmaturity, the founder fish are mated and their progeny analyzed for thepresence of the transgene, such as by Southern blot, PCR and proteinanalysis.

Example 5

The modified lentivirus described above may also be used in gene trapexperiments, such as in zebrafish. As discussed above, this techniqueallows the identification and cloning of a gene that is expressed in aparticular tissue or cell type and/or at a particular time based solelyon its pattern of expression. Zebrafish is an ideal system for genetrapping for several reasons. First, embryonic development occursexternally, allowing for easy manipulation and viewing of the embryos.Furthermore, early stage zebrafish embryos are translucent, and thepigmentation can be further suppressed for several more days byincubating the embryo in a 0.003% solution of 1-phenyl-2-thiourea (PTU).The translucent property of zebrafish embryos facilitates the viewing ofa live fluorescent reporter to identify trapped genes expressed inspatial or temporal patterns of interest.

Self-inactivating lentiviral vectors are engineered to contain a genetrap element consisting of the following sequences: spliceacceptor-IRES-GFP-poly A addition signal. This cassette is called SAIGP.The SAIGP element is inserted in a 3′ to 5′ orientation with respect tothe viral LTR sequences, to prevent inappropriate splicing ortermination of the viral genome during packaging. Zebrafish zygotes areinjected with VSVg-pseudotyped, concentrated SAIGP lentivirus asdescribed above. Fish are raised to sexual maturity and mated. Theprogeny are viewed with a fluorescent microscope, and GFP-expressingindividuals are separated for further analysis. GFP-positive animals arethen analyzed with a confocal fluorescent microscope to determine thespatial and temporal pattern of expression. Messenger RNA is extractedfrom those tissues of the animal that express GFP in the time and placeof interest, and reverse transcription with oligonucleotidescomplementary to GFP yields a cDNA that should contain the sequences ofthe trapped gene that flank the provirus. The recovered cDNA issubcloned into an appropriate bacterial plasmid, and the gene that hasbeen trapped by the SAIGFP provirus is identified by sequencing theupstream regions of the cDNA.

Example 6

Virus particles generated from the FUGW vector were generated asdescribed above. The virus particles were injected using theperivitelline injection method, also described above, into 4 fertilizedrhesus monkey (Macaca mulatta) single cell embryos. Monkey oocytes canbe fertilized directly with sperm or can be fertilized using theintracytoplasmic sperm injection (ICSI) method. Of the four embryosinjected, 2 developed into blastocysts. Both blastocysts were green,evidencing expression of GFP. In the injected blastocysts, cells in thetrophectoder (TE) and the inner cell mass (ICM) both were green.Non-injected control embryos were not green. Transformed embryos aretransferred to host mothers for gestation. After approximately 150 to175 days, a newborn rhesus monkey is delivered which expresses GFPthroughout. Confirmation of the presence of the transgene and expressionin various tissues is carried out as described above.

What is claimed is:
 1. A method of producing a transgenic mousecomprising: transfecting a packaging cell line with: a first retroviralconstruct comprising the R and U5 sequences from a 5′ lentiviral LTR, aself-inactivating 3′LTR, a transgene of interest, and a promoteroperably linked to the transgene of interest; a second retroviralconstruct comprising the HIV-1 packaging vector with the env, nef,5′LTR, 3′LTR, and vpu sequences deleted; and an expression plasmidencoding a pseudotyped envelope glycoprotein; recovering recombinantpseudotyped retrovirus from the packaging cell line; infecting an oocyteor single cell embryo with the recombinant pseudotyped retrovirus,wherein the recombinant pseudotyped retrovirus comprises the R and U5sequences from a 5′ lentiviral long terminal repeat (LTR) and aself-inactivating lentiviral 3′ LTR; and implanting the oocyte oncefertilized or single cell embryo in a pseudopregnant female to produce atransgenic mouse, wherein the pseudotyped retrovirus is integrated intothe genome of the mouse embryo, and wherein the transgenic mouse is ableto pass the transgene of interest to a progeny of the transgenic mousesuch that the progeny expresses the transgene of interest.
 2. The methodof claim 1 wherein said promoter is an internal promoter.
 3. The methodof claim 1 wherein said transgenic mouse expresses the transgene ofinterest.
 4. The method of claim 1 wherein said packaging cell line is a293 cell line.
 5. The method of claim 1 wherein the 5′ LTR sequences arefrom HIV.
 6. The method of claim 1 wherein the self-inactivating 3′ LTRcomprises a U3 element with a deletion of its enhancer sequence.
 7. Themethod of claim 6 wherein the self-inactivating 3′ LTR is a modified HIV3′ LTR.
 8. The method of claim 1 wherein the recombinant retrovirus ispseudotyped with the vesicular stomatitis virus envelope glycoprotein.9. The method of claim 1 wherein the recombinant retrovirus ispseudotyped with a mutant ecotropic envelope protein.
 10. The method ofclaim 1 wherein the promoter is operably linked to the R and U5 5′ LTRsequences.
 11. The method of claim 1, wherein the promoter is a CMVpromoter.
 12. The method of claim 10 wherein the recombinant pseudotypedretrovirus additionally comprises an enhancer operably linked to thepromoter.
 13. The method of claim 12 wherein the enhancer and promoterare CMV sequences.
 14. The method of claim 1 wherein the recombinantpseudotyped retrovirus additionally comprises the woodchuck hepatitisvirus enhancer element sequence.
 15. The method of claim 1 wherein therecombinant pseudotyped retrovirus additionally comprises a tRNA ambersuppressor sequence.
 16. The method of claim 2 wherein the viralconstruct additionally comprises a reporter gene.
 17. The method ofclaim 16 wherein the reporter gene encodes a fluorescent protein. 18.The method of claim 17 wherein said fluorescent protein is greenfluorescent protein.
 19. The method of claim 2 wherein the internalpromoter is a ubiquitous promoter.
 20. The method of claim 19 whereinsaid ubiquitous promoter is selected from the group consisting of theubiquitin promoter, the CMV β-actin promoter, and the pgk promoter. 21.The method of claim 2 wherein the internal promoter is a tissue specificpromoter.
 22. The method of claim 21 wherein said tissue specificpromoter is selected from the group consisting of the lck promoter, themyogenin promoter, and the thy1 promoter.
 23. The method of claim 1wherein infecting an oocyte or single-cell embryo comprises injectingthe recombinant retrovirus between the zona pellucida and the cellmembrane of a mouse oocyte or single-cell mouse embryo.
 24. The methodof claim 1 wherein infecting an oocyte or a single-cell embryo comprisesremoving the zona pellucida from a mouse oocyte or single-cell mouseembryo and incubating the cell in solution containing the recombinantretrovirus.
 25. The method of claim 24 wherein the zona pellucida isremoved by enzymatic digestion.
 26. A method of producing a transgenicmouse comprising the following steps: a) removing the zona pellucidafrom a single-cell embryo; b) contacting the single-cell embryo with apseudotyped retrovirus, wherein the pseudotyped retrovirus comprises theR and U5 sequences from a 5′ lentiviral long terminal repeat (LTR), aself-inactivating lentiviral 3′ LTR, and a transgene of interest; and c)implanting the single-cell embryo in a pseudo-pregnant female to producea transgenic mouse, wherein the transgenic mouse is able to pass thetransgene of interest to a progeny of the transgenic mouse such that theprogeny expresses the transgene of interest.
 27. The method of claim 26wherein the pseudotyped retrovirus is a pseudotyped lentivirus.
 28. Themethod of claim 27 wherein the pseudotyped lentivirus is produced bytransfecting a packaging cell line with: a first retroviral constructcomprising the R and U5 sequences from a 5′ lentiviral LTR, aself-inactivating 3′LTR, a transgene of interest and a promoter operablylinked to the transgene of interest; a second retroviral constructcomprising the HIV-1 packaging vector with the env, nef, 5′LTR, 3′LTR,and vpu sequences deleted; and an expression plasmid encoding apseudotyped envelope glycoprotein.
 29. The method of claim 26 whereinthe single-celled embryo is contacted with the pseudotyped retrovirusfor at least 5 hours.
 30. A method of producing a transgenic mousecomprising injecting a pseudotyped lentivirus into the perivitellinespace of an embryonic cell and implanting the embryonic cell in apseudopregnant female to produce a transgenic mouse, wherein thepseudotyped lentivirus comprises the R and U5 sequences from a 5′lentiviral long terminal repeat (LTR), a self-inactivating lentiviral 3′LTR, and a transgene of interest, and wherein the transgenic mouse isable to pass the transgene of interest to a progeny of the transgenicmouse such that the progeny expresses the transgene of interest.
 31. Themethod of claim 1, wherein the recombinant pseudotyped retroviruscomprises the CMV enhancer/promoter sequence fused to the R and U5sequences from the 5′ HIV LTR, the HIV-1 flap signal, an internalenhancer/promoter, an exogenous gene of interest, the woodchuckhepatitis virus responsive element, a tRNA amber suppressor sequence, aU3 element with a deletion of its enhancer sequence, the chickenβ-globin insulator, and the R and U5 sequences of the 3′ HIV LTR. 32.The method of claim 1 wherein the pseudotyped envelope glycoproteincomprises a VSVg envelope glycoprotein.
 33. A method of producing atransgenic mouse comprising the following steps: transfecting apackaging cell line with: a first retroviral construct comprising the Rand U5 sequences from a 5′ lentiviral LTR, a self-inactivating 3′LTR, atransgene of interest, and a promoter operably linked to the transgeneof interest; a second retroviral construct comprising the HIV-1packaging vector with the env, nef, 5′LTR, 3′LTR, and vpu sequencesdeleted; and an expression plasmid encoding a pseudotyped envelopeglycoprotein; recovering recombinant pseudotyped retrovirus from thepackaging cell line; removing the zona pellucida from a single cellembryo; infecting the single cell embryo with the recombinantretrovirus; and implanting the single cell embryo in a pseudopregnantfemale to produce a transgenic mouse, wherein the pseudotyped retrovirusintegrates into the genome of the mouse embryo such that the transgeneof interest is expressed in a progeny of the transgenic mouse.
 34. Amethod of producing a transgenic mouse comprising the following steps:transfecting a packaging cell line with: a first retroviral constructcomprising the R and U5 sequences from a 5′ lentiviral LTR, aself-inactivating 3′LTR, and a transgene of interest, and a promoteroperably linked to the transgene of interest; a second retroviralconstruct comprising the HIV-1 packaging vector with the env, nef,5′LTR, 3′LTR, and vpu sequences deleted; and an expression plasmidencoding a pseudotyped envelope glycoprotein; recovering recombinantpseudotyped retrovirus from the packaging cell line; infecting thesingle cell embryo with the recombinant retrovirus by injecting apseudotyped lentivirus into the perivitelline space of an embryoniccell; and implanting the single cell embryo in a pseudopregnant femaleto produce a transgenic mouse, wherein the pseudotyped retrovirusintegrates into the genome of the mouse embryo, and wherein thetransgenic mouse is able to pass the transgene of interest to a progenyof the transgenic mouse such that the transgene of interest is notinactivated in the progeny.